An understanding of ion channels is fundamental to the study of many biophysical systems, yet several challenges hinder the ability to study ion channels. While more than 50% of all major drug targets are membrane-associated proteins, and over 400 ion channels have been identified in the human genome, only a small percentage have been cloned and functionally studied. As a result, many experimental methods have been developed for the study of ion channels. Current methods for studying ion channels consist of those that analyze ion channels at the single molecule level and those that analyze them at the ensemble level. Further, methods for studying ion channels can be separated into those that analyze ion channel behavior in synthetic settings, and those that study ion channels in biological or native-like settings.
Conventional methods using native-like or biological settings include traditional patch clamp electrophysiology systems such as those that utilize X. laevis oocytes as a platform for heterologous ion channel expression. Methods using the X. laevis oocyte are typically used to replicate and study large numbers of ion channels.
Conversely, conventional methods that use synthetic settings are typically designed for studying ion channels at the single molecule level. For example, the planar lipid bilayer technique is a method in which synthetic phospholipids are applied to a micrometer-sized opening separating two aqueous compartments, thereby forming a lipid bilayer film. The application of the phospholipids to the opening is described as “painting.” After painting with the synthetic phospholipids, proteoliposomes are added to one or both aqueous compartments, and ion channel activity is then monitored using an amplifier, usually under voltage clamp conditions.
Although use of the planar lipid bilayer method for the research of ion channels has been widespread, there are several drawbacks associated with this technique and other similar methods. For example, the planar lipid bilayer method is technically demanding, and is neither robust nor scalable. Further, the lipid bilayers produced via this technique are delicate, and thus susceptible to rupture.
More recently, a droplet interface bilayer (DIB) method has been developed as an alternative to the planar lipid bilayer method. This DIB method comprises the insertion of aqueous droplets into a bulk/oil lipid phase. This DIB method, however, requires both operation in the microfluidic regime and micromanipulation. Because manipulation of nanoliter volume droplets is technically demanding and micromanipulators are bulky, expensive and preclude scalable DIB assembly, this alternative method possesses many of the drawbacks as the planar lipid bilayer method. Importantly, while improvements to the DIB assembly have been described, including droplet dielectrophoresis and digital microfluidics, these strategies require expensive, complicated and not readily accessible clean room device fabrication, iterative rapidly prototypable device design is not possible.
Although there are several contributing factors as to why so few ion channels have been studied, two predominant factors are the technical expertise needed to perform the studies and the high expense of studying ion channels in synthetic environment—drawbacks that remain a concern for conventional methods such as the planar lipid bilayer technique and the previous DIB method. Thus, there is a need for a method for producing lipid bilayers that is inexpensive, scalable, and does not require a high level of technical expertise on the part of the performer, in order to more efficiently study ion channels.